Project Summary ACIS, LLC proposes a conceptually novel cellular heterogeneity assay, based on confocal Raman spectrometry and Biomolecular Component Analysis (BCA). The goal of Phase II is to implement a BCA toolbox in a commercial confocal micro- Raman instrument for quantitative assessment of cellular heterogeneity and potential quantitative classification of cellular states based on macromolecular compositions in specific organelles, a single cell assay developed during the Phase I, Phase II focuses on bringing this new device to the market. This project is motivated by the fact, that there is no currently any commercial tool, which provides direct probing of local biomolecular concentration in live cells. At the same time, this tool is extremely valuable for solving a number of problems, which require determination of quantitative markers for different cellular states (i) in diseased cell population (cancer-non-cancer, different cancer stages etc), (ii) during cell-drug interaction, and (iii) in the intracellular processes (apoptosis, proliferation, differentiation, etc). The Phase I research and development of this assay have met stated technical milestones. In particular, it is found that the BCA potential for characterization of cellular heterogeneity significantly exceeds that of the standard math tool of Principal Component Analysis routinely used for analysis of Raman spectra,. The specific aims of this proposal are: (1) Adaptation of a Thermo-Fisher micro-Raman system DXR2 model to meet requirements for BCA applications; (2) Development of Biomolecular Component Analysis Software package in C++ and/or Java and incorporation to DXR2 model of Thermo-Fisher micro-Raman system; (3) Validation of the BCA toolbox integrated with Thermo-Fisher micro-Raman system DXR2 model for ongoing biomedical research (a) Comparative analysis of heterogeneity between IDH- mutated and WT-IDH glioma cells and assessment of cell-drug interaction in IDH-mutant cells in collaboration with NCI, (Bethesda, MD) (b) Molecular profiling of cellular organelles for categorization of Prostate Cancer with RPCI (Buffalo, NY); (4) Pre-market evaluation of the Thermo-Fisher DXR2 micro-Raman system, equipped by the BCA toolbox. The outcome of this project will be a commercial confocal micro-Raman system with an implemented BCA toolbox, along with customized software for quantitative assessment of cellular heterogeneity, which can be used for a broad range of biomedical applications in many sectors such as clinical labs, Biomedical cellular Research labs, Pharmaceutical industries, National Cancer Institutes , etc.